Somatostatins are ubiquitous polypeptides known to affect basic biological processes such as growth, development, metabolism, and cell differentiation in vertebrates. Somatostatin was first isolated as a 14-amino acid peptide from ovine hypothalamus and found to inhibit the release of growth hormone from the pituitary gland (Brazeau et al., Science, 179, 77-79 (1973)). Since then somatostatins have been isolated from representatives of nearly every major group of vertebrates examined to date, from jawless fish to mammals (Conlon et al., Regul Peptides, 69, 95-103 (1997)). Somatostatins have been found broadly in the central (e.g., cerebral cortex, cerebellum, pineal, olfactory lobe, hypothalamus, spinal cord) and peripheral nervous systems, gastrointestinal tract (e.g. salivary glands, stomach, intestine), urogenital tract (e.g., bladder, prostate, collecting ducts of the kidney), pancreatic islets, adrenal glands, thyroid tissue, and placenta as well as in cerebral spinal fluid, blood, and saliva (Reichlin, xe2x80x9cSomatostatin,xe2x80x9d Brain peptide, Krieger et al., eds., John Wiley and Sons, New York, pp. 711-752(1982); Gerich, xe2x80x9cSomatostatin and analogues,xe2x80x9d Diabetes mellitus: Theory and practice, Ellenberg et al., eds., Medical Examinations, New York (1983); Wass, xe2x80x9cSomatostatin,xe2x80x9d Endocrinology, DeGroot, ed., W B Saunders, Philadelphia, Pa. (1989); Patel, xe2x80x9cGeneral aspects of the biology and function of somatostatin,xe2x80x9d Basic and clinical aspects of neuroscience, Weil et al., eds., Springer-Verlag, Berlin (1992)). In neurons and cells, somatostatins are often co-localized with other factors (e.g., norepinephrine, CCK, neuropeptide-Y, CGRP, GABA, VIP, substance P) (Gibbins, xe2x80x9cCo-existence and co-function,xe2x80x9d The comparative physiology of regulatory peptides, Holmgren, ed., Chapman and Hall, London/New York (1989)).
Somatostatins also possess a vast diversity of physiological actions. In addition to secretotropic effects (including the effect on growth hormone secretion for which the family was named), somatostatins have been reported to have neurotropic and myotropic effects as wells as effects on transport, metabolism, growth, differentiation, and modulation of functional development. It should be noted that there is overlap between and among these somewhat arbitrary classes of action. For example, the inhibition of growth hormone secretion clearly affects growth and the inhibition of insulin secretion clearly affects metabolism. At the same time, the inhibition of growth hormone also impacts metabolism while the inhibition of insulin has ramifications on growth (Norman and Litwack, Hormones, Academic Press, New York (1997)). In addition to such actions which result in physiological xe2x80x9ccross talk,xe2x80x9d somatostatins also have direct effects on the various classes of action. For example, somatostatins have been shown to affect growth (e.g., proliferation) and intermediary metabolism (e.g., lipolysis) directly in target cells (Patel, xe2x80x9cGeneral aspects of the biology and function of somatostatin,xe2x80x9d Basic and Clinical Aspects of Neuroscience, Weil et al., eds., Springer-Verlag, Berlin (1992); Sheridan, Comp. Biochem. Physiol., 107b, 495-508 (1994)). Considering these various roles, somatostatins may be of considerable importance in various diseases including neuroendocrine tumors, diabetes mellitus, epilepsy, Alzheimer and Huntington Diseases, and AIDS (Lamberts et al., Endocrine Rev., 12, 450-482 (1991); Patel et al., Life Sci., 57, 1249-1265 (1995)).
Most somatostatins appear to be synthesized as a long chain prepropeptide, which can be subsequently processed to yield a propeptide (typically ranging in size from 25-28 amino acids) and, in some cases, further processed to yield a peptide of about 14 amino acids. This differential processing introduces considerable molecular heterogeneity into somatostatins. It is believed that in mammals, differential processing of the transcription product of a single gene may account for the tissue-, organ- and cell-specific activities of various somatostatins. The bioactivity of secreted somatostatins is mediated by cell-surface somatostatin receptors which likely differentiate among the various forms of somatostatin present in an organism. The molecular heterogeneity of somatostatins appears to be even greater in some non-mammalian organisms. Fish and some other non-mammals, for example, may possess several somatostatin genes, each of which may be differentially processed.
Notwithstanding the heterogeneity that characterizes the longer chain preprosomatostatins and prosomatostatins, the somatostatin tetradecapeptide SS-14 (Ala-Gly-Cys-Lys-Asn-Phe-Phe-Trp-Lys-Thr-Phe-Thr-Ser-Cys; SEQ ID NO:1), present at the C-terminus of the longer forms, is completely conserved among such mammals as monkeys, rats, cows, sheep, chickens and humans. Somatostatins found in both mammals and non-mammals typically contain the C-terminal SS-14 sequence (SEQ ID NO:1). Non-mammals, however, frequently express additional somatostatins that contain variant C-terminal tetradecapeptides with substitutions at one or more sites, such as [Tyr7, Gly10]-SS-14 (SEQ ID NO:2). This alternative somatostatin peptide has modifications at positions 7 and 10 when compared to the mammalian sequence [Phe7, Thr10]. Somatostatins that contain the xe2x80x9cmammalianxe2x80x9d-type 14-mer sequence (SEQ ID NO:1) at the C-terminus are considered to be part of the xe2x80x9cSS-Ixe2x80x9d family, whereas those that contain a 14-mer sequence having the [Tyr7, Gly10] modification (SEQ ID NO:2) are considered to be part of the xe2x80x9cSS-IIxe2x80x9d family.
In mammalian systems, somatostatin is secreted into the blood and is vascularly active. Different cells can synthesize different versions of the polypeptide. Secreted somatostatin is also known to have a local paracrine activity. There are a number of human diseases (e.g., growth disorder, diabetes, and several neurological disorders) that may be treated with somatostatin analogs. Also, some conditions (e.g., tumors) result from overproduction of somatostatin, and there is no known somatostatin antagonist for treatment of such disorders. New somatostatin analogs (both agonists and antagonists) that have the potential to treat these and other human diseases would be a welcome addition to current therapeutic strategies.
The invention provides novel somatostatin polypeptides that contain amino acid sequences found in Oncorhynchus mykiss preprosomatostatin I (PPSS-I; SEQ ID NO:3) and/or Oncorhynchus mykiss preprosomatostatin IIxe2x80x3 (PPSS-IIxe2x80x3; SEQ ID NO:15). Also provided are bioactive analogs and subunits of the somatostatin polypeptides of the invention. Preferred somatostatin polypeptides include polypeptides having at least one amino acid sequence selected from the group consisting of SEQ ID NOS:1, 2, 3, 4, 5, 6, 7, 9, 10, 11, 12, 13, 15, 16, 17, 18, and 19, and bioactive analogs and subunits thereof.
Polynucleotides encoding somatostatin polypeptides of the invention and/or bioactive analogs and subunits thereof, as well as those that are substantially complementary thereto, are also provided.
The invention further provides a method for identifying a modified somatostatin polypeptide. The amino acid sequence of a somatostation polypeptide of the invention is aligned with the amino acid sequence of a reference somatostatin polypeptide, preferably a mammalian somatostatin polypeptide, and at least one site or region of differing amino acid sequence is identified. Either the somatostatin polypeptide of the invention or the reference somatostatin polypeptide is then modified to incorporate at least one amino acid substitution, insertion, or deletion from the analogous site or region in the other somatostatin polypeptide to yield the amino acid sequence of a modified somatostatin polypeptide. Optionally, the method further includes synthesizing the modified somatostatin polypeptide and assaying the modified somatostatin polypeptide for biological activity. Biological activity is preferably determined by determining whether the modified somatostatin polypeptide binds to a human somatostatin receptor molecule or inhibits the binding of a natural ligand of the human somatostatin receptor molecule. Preferably, the modified somatostatin polypeptide identified according to the method of the invention is a somatostatin agonist or antagonist.
Also provided by the invention is a fusion polypeptide, wherein an N-terminal somatostatin region is fused (i.e., covalently linked) to a selected C-terminal region. The N-terminal somatostatin region includes one or more first amino acid sequences that contain an isoform or isoform fragment of PPSS-I and/or PPSS-IIxe2x80x3 as described herein, or portion thereof. The C-terminal region contains a second amino acid sequence that preferably encodes a bioactive peptide moiety.